Project Summary/Abstract Progress in glycomics, the systematic study of the structure and function of glycans, lags far behind that of genomics and proteomics due to the more complex structures of glycans, whose biosynthesis is posttranslational and not template-driven. A major limitation in glycomics is the absence of powerful amplification technologies such as PCR, recombinant technology and automated chemical synthesis, which limits the availability of biologically relevant glycans for functional studies. To address this limitation, we developed a novel technology, oxidative release of natural glycans (ORNG), which uses simple oxidation by sodium hypochlorite (household bleach) to degrade peptides and nucleic acids and release bleach-resistant glycans from the glycoconjugates of natural sources such as animal/plant tissues. We have demonstrated that unlike current methods for releasing glycans, ORNG is a scalable process which has allowed us to generate unprecedented amounts of glycans from up to 20 kg of animal tissue. Thus, the absence of glycan amplification technology can be addressed by increasing the amount of starting material, which results in large quantities of released, biologically relevant glycans. Glycan vendors manufacture N-glycans using non- scalable methods resulting in limited amounts of material and high costs (20 g packages for $200 to $500). Our scalable process will allow us to generate gram quantities of high-mannose, paucimannose, and complex- type N-glycans, and the economy realized by the large scale will allow us to provide mg quantities of N-glycans at <10% of current costs making these glycans available to non-specialized laboratories in mg quantities for functional studies. We have demonstrated the utility of our process for manufacturing mg quantities of N- glycans, which are not currently available in these quantities. However, ORNG is unique in that it releases all classes of glycans in a single, rapid reaction, and we have currently optimized it for release of N-glycans to demonstrate feasibility. Here, we are requesting funds to develop a streamlined ?downstream process? for purification of all classes of glycans including glycosaminoglycans, O-linked glycans and glycosphingolipid- linked glycans from a single oxidation run. Vendors of O-linked glycans and glycosphingolipid-linked glycans generally manufacture them by chemical or chemo-enzymatic synthesis, which is expensive and limited to single products that are selected based on our limited information of what structures exist in nature. Since ORNG uses kg quantities of natural products as starting material, the resulting glycans will represent a combinatorial library of the source natural product containing not only the known, common glycans, but also minor glycans that have not been previously detected. This project will result in a robust manufacturing process for purified, natural N-, O-, GSL-linked glycans that we can offer at affordable prices. In addition, we will manufacture the corresponding shotgun glycan microarrays based on these ?combinatorial? libraries that can be interrogated with biologically relevant GBPs to discover novel glycans with unique functions. 1